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　We previously reported that embryonic stem (ES) cells cultured on M15 cells, a mesoderm-derived supportive cell line, were efficiently differentiated towards an endodermal fate, finally adopting the specific lineages of various digestive organs such as the pancreas and liver. We show here that the endoderm-inducing activity of M15 cells is in part mediated through the extracellular matrices, and that laminin α5 is one of the crucial components. In an attempt to establish a feeder-free ES-cell procedure for pancreatic differentiation, we used a synthesized basement membrane (sBM) substratum using an HEK293 cell line stably expressing laminin-511. On the sBM, mouse ES or induced pluripotent stem (iPS) cells sequentially differentiated into the definitive endoderm, pancreatic progenitor cells, and then insulin-expressing pancreatic b -cells in vitro. Knockdown of ES cells with integrin β1 ( Itgb1 ) reduces differentiation towards pancreatic cells.Heparan sulfate proteoglycan 2 ( HSPG2 ) knockdown and heparitinase treatment synergistically decreased the number of Pdx1 -expressing cells. These findings indicate that components of the basement membrane have an important role in the differentiation of definitive endoderm lineages. This novel procedure will be useful for the study of pancreatic differentiation of ES or iPS cells and the generation of potential sources of surrogate cells for regenerative medicine.

 

 

Figure. Differentiation signals from the BM.

A schematic of the differentiation signals from the BM. One of the BM components, Laminin α5, interacts with integrin β1, leading to pancreatic differentiation. HSPG2 in BM mediated pancreatic differentiation. Moreover, various kinds of signaling molecules are trapped in the heparan sulfate chain of HSPG2 or other HSPGs. These molecules directly and/or indirectly induce pancreatic differentiation.